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The Roles of Essential Proteins Prp6 and Dib1 During Pre‐mRNA Splicing in Saccharomyces cerevisiae
Author(s) -
Embry Addie,
Jameson Danielle,
Turner Caitlyn,
Maeder Corina
Publication year - 2020
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2020.34.s1.03574
Subject(s) - spliceosome , rna splicing , snrnp , prp24 , small nuclear rna , sr protein , protein splicing , biology , small nuclear ribonucleoprotein , minor spliceosome , genetics , exonic splicing enhancer , ribonucleoprotein , microbiology and biotechnology , rna , gene , non coding rna
Pre‐mRNA splicing is a conserved biological process essential to all eukaryotes. Splicing defects lead to a variety of disorders in humans including Retinitis Pigmentosa and Burn‐McKeown Syndrome, defining the importance of splicing in health science fields. Pre‐mRNA splicing requires complex machinery composed of five small nuclear RNAs (snRNAs) complexed with numerous proteins that form five small nuclear ribonucleoproteins (snRNPs). Spliceosome assembly occurs over several dynamic steps to form a pre‐catalytic B complex before splicing reactions can occur (Will, et al. 2011; Plaschka, et al. 2017). Splicing cannot occur until the pre‐mRNA can interact with the Prp8 protein and the U5 snRNA in the catalytic center of the spliceosome. Analysis of cryo‐EM structures reveal that the essential proteins Dib1 and Prp6 in the B complex block pre‐mRNA from interaction with the catalytic site. Dib1, a 17‐kDa protein with a thioredoxin‐like (TRXL) domain, sits at the center of the catalytic site (Plaschka, et al. 2017). Although small, Dib1 contains distinct features that includes a solvent‐exposed hydrophobic groove which appears to interact with the N‐terminus of Prp6, a large, 102‐kDa U4/U6.U5 triple‐snRNP protein (Reuter, et al. 1999; Plaschka et al. 2017). Studies of S. pombe and human splicing proteins demonstrated interactions between Prp6 and Dib1 orthologs, and deletion of at least the first 50 amino acids of the Prp6 N‐terminal region halted pre‐mRNA splicing in S. pombe (Liu, et al. 2006; Lutzelberger, et al. 2010). Prp6 and Dib1 must depart the spliceosome before splicing can occur. The interactions between these proteins and the catalytic site of the B complex may serve to stabilize the spliceosome and prevent premature pre‐mRNA splicing. Interactions between Prp6 and Dib1 may also mediate assembly of the spliceosomal B complex. We previously reported that a temperature‐sensitive ( ts) mutation in the Dib1 hydrophobic pocket impaired pre‐mRNA splicing, and preliminary assembly assays run on native polyacrylamide gels suggest the Dib1 mutation affects spliceosome assembly. Additionally, we reported that a N‐terminal deletion of Prp6 also halted splicing. Here, we will report new ( ts ) growth mutants in the N‐terminus of Prp6 that may interact with the Dib1 hydrophobic groove and determine if they inhibit splicing. We will also report continued studies of the effects of existing Prp6 and Dib1 mutants and how they affect splicing and spliceosome B complex assembly. Support or Funding Information NIH‐NIGMS R15GM120720 and The Welch Foundation